Aplastic Anemia With Thrombosis Following the Administration of Immunosuppressant and Thrombopoietin Receptor Agonist (TPO-RA)

Thrombopoietin receptor agonist (TPO-RA) is effective for aplastic anemia (AA) and idiopathic thrombocytopenic purpura (ITP). However, the risk of thrombosis during ITP treatment with TPO-RA is higher than without TPO-RA. It is unclear whether TPO-RA increases the risk of thrombosis in patients with AA. We report a case of a 66-year-old female with severe AA having paroxysmal nocturnal hemoglobinuria (PNH) clones in the peripheral blood who developed ischemic colitis after three days of starting eltrombopag. Contrast-enhanced computed tomography showed ischemic colitis and contrast enhancement defect in the left atrial appendage, which indicated a thrombus in the heart. Stopping eltrombopag and providing supportive care improved her symptoms, and her blood cell counts gradually increased. Thrombosis should be considered when TPO-RA is administered during the immunosuppressive treatment of AA.


Introduction
In treating aplastic anemia (AA), thrombopoietin receptor agonists (TPO-RAs), such as eltrombopag and romiplostim, have improved response rates when combined with immunosuppressive therapy [1,2].Eltrombopag is a small oral molecule that binds to the TPO receptor and promotes hematopoiesis and platelet production.TPO receptor is expressed in hematopoietic stem cells, precursor cells of the megakaryocytic lineage, megakaryocytes, and mature platelets [3].TPO-RA is not directly associated with exacerbating platelet functions such as platelet aggregation.However, clinically, especially in treating immune thrombocytopenia (ITP), TPO-RA administration increases the risk of arterial and venous thrombotic events during the platelet recovery period and periods of low platelet count, according to metaanalysis [4].Cases of thrombosis and ischemic colitis during treatment with eltrombopag for AA are rare, but they are considered significant complications that warrant attention and reporting.

Case Presentation
A 66-year-old female had a complaint of fatigue around June 2019.She was referred to our hospital in August 2019 after her family doctor found that her white blood cell count was 2600/µL, her hemoglobin level was 8.6 g/dL, and her platelet count was 36000/µL.She had a history of hypertension, chronic heart failure, and atrial fibrillation and was taking antihypertensive medications and warfarin.She was admitted to our hospital in September 2019 for a further evaluation of her pancytopenia.On admission, she was clearly conscious, with a temperature of 36.4°C, a pulse of 90 beats/min, a blood pressure of 116/76 mmHg, and a percutaneous blood oxygen saturation of 98% (room air).She had conjunctival pallor and no abdominal tenderness.Blood tests showed pancytopenia and coagulation system tests showed prolonged prothrombin time due to warfarin's anticoagulation for atrial fibrillation (Tables 1-4).We adjusted the warfarin dosage so that the prothrombin time-international normalized ratio (PT-INR) remained in the therapeutic range, i.e., around 2 (1.6-2.6), before the onset of ischemic enteritis after hospitalization.

Normal range
White blood cells 2.4×10     Based on her peripheral blood data and MRI findings of the bone marrow in her spine, which showed a high degree of fatty marrow with the high-intensity signal on TI-weighted images and a low-intensity signal on short tau inversion recovery (STIR) images, she was suspected of having AA.A bone marrow biopsy image showed hypoplastic bone marrow (Figure 1).Electrocardiography showed atrial fibrillation, but transthoracic echocardiography showed no evidence of intracardiac thrombosis.High-sensitivity paroxysmal nocturnal hemoglobinuria (PNH) blood cell analysis using flow cytometry showed 0.098% of type III PNH neutrophils, which were negative for fluorescent-labeled inactive toxin aerolysin (FLAEAR), and 0.20% of type III PNH erythrocytes were negative for CD55 and CD59 (Figures 2a, 2b).

FIGURE 1: Bone marrow pathology.
Hypoplastic marrow with some lymphocytes was observed.There were few megakaryocytes in the microscope field of vision.We diagnosed her with severe aplastic anemia based on peripheral blood data and bone marrow pathology using Camitta criteria [4].Figure 3 shows her clinical course since the start of treatment.Aplastic anemia was treated with antithymocyte immunoglobulin (ATG), immunosuppressive therapy with cyclosporine (CSP), and steroids.We administered 200 mg of thymoglobulin as ATG and 125 mg of methylprednisolone intravenously for five days and initiated oral administration of 300 mg (5 mg/kg/day) of CSP.Then methylprednisolone was tapered off.Fever, allergic symptoms, and serum sickness-like symptoms associated with the use of rabbit-derived ATG were not seen due to the concomitant use of high-dose steroids.On the 18th day after the start of immunosuppressive therapy, the patient complained of constipation and abdominal pain, and a contrastenhanced CT scan was performed, but there were no significant findings.On the 15th day of immunosuppressive therapy, eltrombopag 75 mg was started orally, and on the third day of eltrombopag (18th day of immunosuppressive therapy), abdominal distention and abdominal pain re-appeared, and there was marked increase in intestinal gas and melena appeared.She developed hemodynamic instability, and her systolic blood pressure dropped to 70 mmHg.We transfused her with red blood cells and platelets and managed her circulatory failure.Her white blood cell count was 2600/µL, her hemoglobin level was 6.1 g/dL, and her platelet count was 13000/µL when her condition suddenly changed.Abdominal and chest contrastenhanced CT showed edema of the colon and intestinal tract, opacity of the surrounding fatty tissue, and air in the portal vein, which suggested ischemic enteritis, and filling defect image in the left auricle, which appeared to be a thrombus (Figures 4a-4f).During the thrombotic event, the D-dimer increased up to 7.9 μg/mL (normal range: 0.0-1.0μg/mL), which indicates that thrombosis had occurred, and it took two months for the D-dimer to return to the normal range.The PT-INR at the time of the ischemic enteritis was 2.56 and was controlled within the therapeutic range.In this case, liver and renal function were routinely monitored with blood tests before and after the thrombotic event.However, no hepatic or renal dysfunction due to TPO-RA administration was observed during the observation period.The platelet count remained around 10000/µL during the clinical course, and we transfused platelets when the platelet count fell below 10000/µL.After the onset of gastrointestinal symptoms, she was made nil per os, given broad-spectrum antibiotics, and discontinued eltrombopag, CSP, and warfarin.Her general condition gradually improved, and she resumed eating three days after the onset of ischemic enteritis.CSP was resumed at 100 mg/day on day 29 after the start of immunosuppressive therapy, and eltrombopag was resumed at 25 mg/day on day 51.She did not receive any transfusion after platelet transfusion on day 50 after the start of immunosuppressive therapy, and she resumed taking warfarin for atrial fibrillation on day 40 of immunosuppressive therapy.She was discharged from the hospital 68 days after the start of immunosuppressive therapy and is still being followed in the outpatient.There was no evidence of thrombocytopenia or new thrombus, and a CT scan 10 months after the start of immunosuppressive therapy showed that the thrombus in the heart had been obscured.

Discussion
Aplastic anemia is a rare hematologic disease and is a hematopoietic disorder caused by immunologic mechanisms to hematopoietic stem cells.Clinically, it is characterized by pancytopenia, with associated fatigue, bleeding, and infection as the main symptoms.The severity of AA should be assessed according to the Camitta criteria based on marrow cellularity, neutrophils count, platelet count, and reticulocyte count.
We diagnosed this patient with severe aplastic anemia based on marrow cellularity <25%, platelet count <20000/µL, and reticulocyte count <60000/µL.The recommended primary treatment for severe aplastic anemia is ATG+ CSP+/-eltrombopag in patients over 40 years [2,5].Complications of this treatment, particularly associated with ATG, include allergic reactions due to heterologous proteins, transient cytopenia associated with antibody reactions, infections due to immunodeficiency, and secondary clonal abnormalities.In the present case, transient thrombocytopenia and neutropenia developed after ATG administration, resulting in increased platelet transfusions and filgrastim administration as granulocyte colony-stimulating factor (G-CSF) increased.High-dose steroids in combination with ATG and G-CSF may have contributed to the development of thrombosis in this patient.Eltrombopag is an oral TPO-RA associated with thrombosis and hepatic and renal dysfunction, stimulating the megakaryocyte's hematopoiesis.Eltrombopag has been shown to effectively increase platelets in AA and ITP and myelodysplastic syndrome (MDS) by stimulating megakaryopoiesis.Endogenous TPO binds to the extracellular domain of TPO receptors.In contrast, eltrombopag binds to the transmembrane domain of TPO receptors on blood cells and stimulates platelet production via the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway [3,6].Although eltrombopag is not directly involved in platelet activation in vitro and in vivo, it has been reported to increase the risk of thrombosis in clinical practice, especially in treating ITP [6].A meta-analysis showed that thrombosis occurred in 3.69% of patients treated with TPO-RA, significantly more than in controls not treated with TPO-RA.The reason why TPO-RA causes thrombosis is unknown.The mechanism of TPO-RA-induced thrombosis is that TPO-RA promotes platelet aggregation by increasing p-selectin, an adhesion molecule on the platelet surface [7].A search of case reports of thrombosis associated with AA, not limited to TPO-RA, revealed 12 cases, including the present case (Table 5).

Estimated cause of thrombosis Vessel/organ of thrombosis Platelet count (/μL) Treatment for thrombosis Outcome References
NA (not available) Splenic artery, pulmonary vein Absent None Death [8] Metenolone enanthate Coronary artery 40000 None Improved [9]   Oxymetholone Coronary artery 10000 None Improved [9]   Plasminogen Tochigi Cerebral artery, splenic artery, renal artery 3000 Warfarin Improved [10]   Antiphospholipid syndrome Cerebral venous sinus 22000 Aspirin, mephylprednisolone, CSP Improved [11]   Danazol Cerebral venous 90000 Low molecular weight heparin Improved [12]   Platelet transfusion Coronary stent 12000 Balloon angioplasty Improved [13]   PNH clone Superior mesenteric vein, portal vein 29000 Warfarin Improved [14]   CSP Cerebral venous sinus thrombosis 55000 Enoxaparin Improved [15]   Eltrombopag Venous thrombus NA NA NA [16] Eltrombopag  The presumed causes in the literature were anabolic hormone administration in three cases [9,12], congenital coagulation gene abnormality in one case [9], antiphospholipid antibody syndrome in one case [11], platelet transfusion in one case [13], PNH blood cell involvement in one case [13], and CSP in one case [15].We believe that eltrombopag was one of the causes in this case, but the presence of PNH blood cells and the fact that the patient was receiving CSP and platelet transfusion may also have been involved in the thrombus development.In the present case, atrial fibrillation induced a thrombus in the left ear, which may have led to ischemic enterocolitis, even though the patient's PT-INR was well within therapeutic range with warfarin and rather prolonged due to the interaction of CSP.Although observation of the colonic mucosa by endoscopy is essential for diagnosing hematogenous enteritis, colonoscopy was not performed in this case because of thrombocytopenia.The patient had comorbidities such as atrial fibrillation and constipation prior to the onset of aplastic anemia, which may have been associated with the development of this thrombotic event.It is possible that the decreased blood flow to the colonic mucosa associated with increased abdominal pressure during defecation was a contributing factor to ischemic enteritis.It was unlikely that malignancy caused the thrombosis since no obvious malignant complications were found on CT scan examination or other screening.Since there was no activated partial thromboplastin time (APTT) prolongation, we considered it unlikely that antiphospholipid antibody syndrome was the cause.As for thrombus formation, eltrombopag may have increased p-selectin and other substances on the platelet surface, and platelet transfusion and other factors may have also contributed to platelet aggregation.Alternately, PNH blood cells, such as platelets, monocytes, and neutrophils without the complement regulator CD59, promote thrombus formation and may cause thrombotic events in this case [17,18].Regarding the platelet count at the time of thrombosis, in all previous reports, including the present case, thrombosis occurred with a platelet count lower than the normal range.However, some cases occurred after platelet transfusion or during platelet count recovery.The patient was treated for aplastic anemia with G-CSF, which has been reported to act directly to aggregate platelets [19].It is unclear to what extent G-CSF affects thrombosis when platelet counts are low, but the combination of factors, TPO-RA, G-CSF, steroid administration, and PNH blood cells might act synergistically on thrombogenesis.After the thrombotic event had calmed down, eltrombopag was resumed at a low dose of 25 mg, and the dose was not increased for a long time.After that, the thrombosis did not recur.The low dose of eltrombopag did not cause the recurrence of thrombosis, and the absence of other concomitant medications, such as G-CSF or steroids, did not cause thrombosis.

Conclusions
TPO-RAs, such as eltrombopag and romiplostim, are now widely used for AA.We must remember that thrombotic complications can occur during platelet recovery and when platelet counts are low.When administering these agents, it is essential to monitor platelet counts and the coagulation system regularly, with attention to atrial fibrillation and other thromboembolic risk factors, and to decide whether to continue or discontinue anticoagulation therapy.The treatment of patients with AA who develop thromboembolism and pancytopenia after TPO-RA therapy can be challenging; however, with appropriate supportive care, thromboembolism and AA can be controlled.

FIGURE 4 :
FIGURE 4: Abdominal and chest contrast-enhanced CT.CT scan showed ischemic colitis of the colon, free air in the liver portal vein (a), thickening of the gut wall in the transverse plane (b), and thickening of the gut wall in the coronal plane (c).CT scan of the chest showed complete enhancement of the left atrial appendage 10 days before the day of colitis (d), contrast enhancement defect on the day of colitis (e), and recovered enhancement of the left atrial appendage 10 months after the day of colitis (f).